The majority of human cancers have lost the function of tumor suppressor genes (LKB1, PTEN, TSC1/2) controlling energy metabolism on the Akt-mTOR pathway or have hyperactivity of oncogenes (PI3kinase, Akt) on this same pathway. We have found that drugs that inhibit the second folate-dependent enzyme on the de novo purine biosynthesis pathway cause the accumulation of the substrate for that reaction, ZMP, and that this endogenously synthesized ZMP activates AMP-dependent protein kinase. Compounds that activate AMP kinase hava potential as antitumor agents since this enzyme acts as a key regulator of TORC1 on this pathway, restoring control in tumor cells with these genetic changes. This project proposes to study inhibitors of purine synthesis at the biochemical and molecular genetic levels to determine the mechanism of these effects and the utility of these inhibitors specifically to treat cancers that have genetic defects in the PI3kinase-mTOR pathway. The enzymology and cell biology of the lead compounds would be studied, and more potent inhibitors of the AICART formyltransferase reaction would be designed as targeted therapeutic agents PUBLIC HEALTH RELEVANCE: The majorities of human cancers have lost the function of tumor suppressor genes controlling energy metabolism on the Akt-mTOR pathway or have hyperactivity of cancer-causing genes on this pathway. We have found that drugs that inhibit a folate- dependent enzyme on the purine biosynthesis pathway cause the accumulation of a metabolite, ZMP, that can reactivate the AMP-dependent protein kinase that regulates this pathway, restoring control in tumor cells with these genetic changes. This project proposes to study this effect at the biochemical and molecular genetic levels and to develop potent inhibitors of this folate-dependent enzyme.